IL-2 stimulated mouse spleen cells were used as a source of mouse chromosomes for analysis and sorting of the C57BL/6J mouse Y chromosome. The DNA content of this mouse Y chromosome is approximately 94.7 megabases, making it similar in size to human chromosome 16, and significantly larger than previous estimates. Two small insert Y chromosome-specific libraries, ideal for sampling Y chromosome sequences were constructed. Clone specificity was confirmed. From the characterized clones were developed 15 male-specific PCR assays, and 19 male-female PCR assays likely to originate from the pseudoautosomal region or other areas of X-Y or autosome-Y homology. (Genomics, 48, 304-313, 1998)